Design of 16S rRNA gene primers for 454 pyrosequencing of the human foregut microbiome
Design of 16S rRNA gene primers for 454 pyrosequencing of the human foregut microbiome
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DOI：
10.3748/wjg.v16.i33.4135
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发表时间：
2010-09-07
影响因子：
4.3
通讯作者：
Pei, Zhiheng
中科院分区：
文献类型：
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作者：
Nossa, Carlos W.;Oberdorf, William E.;Pei, Zhiheng
目的：设计和验证宽范围的16S rRNA引物，用于高通量测序对人类前肠微生物群分离的细菌进行分类。方法：利用Sanger测序从口腔、食管和胃微生物组中获得的16S rRNA基因序列构建前肠微生物组数据集，这些基因序列代表了219种细菌。评估的候选引物来自欧洲rRNA数据库。为了评估序列长度对分类准确性的影响，我们通过修剪全长序列创建了不同长度的16S rRNA基因。选择跨越不同高变区的序列来模拟使用可能的引物对获得的扩增子。利用Ribosomal Database Project （RDP）的在线软件将这些序列与全长16S rRNA基因进行分类准确性比较。利用RDP 16S rRNA数据库评估引物集的通用性，该数据库包含36门433306个16S rRNA基因。结果：在大肠杆菌165 rRNA基因中，从碱基28对应的位置往下游截断100个核苷酸（nt）导致219条序列中有87条（39.7%）被误分类，而截断到350个核苷酸的序列中只有29条（13.2%）被误分类。在165 rRNA基因各区域350个nt的序列中，使用343F/798R引物产生的扩增子反读对分类的影响最小（8.2%）。相比之下，截断到900 nt模仿单次Sanger读取错误分类了5.0%的219个序列。343F/798R扩增子在物种水平上准确定位了219个序列中的91.8%。根据食道数据集中物种的丰度加权，343F/798R扩增子产生了相似的分类精度，而物种覆盖率没有显著损失（92%）。将343F/798R引物修饰为347F/803R，提高了其在前肠物种中的通用性。假设典型的聚合酶链反应可以容忍引物和模板之间的2次错配，则修饰后的347F和803R引物应该能够退火RDP数据库中所有16S rRNA基因的98%和99.6%。结论：347F/803R是最适合前肠16S rRNA基因分类的引物，同时也具有通用性，适用于其他复杂微生物的分析。(C) 2010年白石登。版权所有。
AIM: To design and validate broad-range 16S rRNA primers for use in high throughput sequencing to classify bacteria isolated from the human foregut microbiome.METHODS: A foregut microbiome dataset was constructed using 16S rRNA gene sequences obtained from oral, esophageal, and gastric microbiomes produced by Sanger sequencing in previous studies represented by 219 bacterial species. Candidate primers evaluated were from the European rRNA database. To assess the effect of sequence length on accuracy of classification, 16S rRNA genes of various lengths were created by trimming the full length sequences. Sequences spanning various hypervariable regions were selected to simulate the amplicons that would be obtained using possible primer pairs. The sequences were compared with full length 16S rRNA genes for accuracy in taxonomic classification using online software at the Ribosomal Database Project (RDP). The universality of the primer set was evaluated using the RDP 16S rRNA database which is comprised of 433306 16S rRNA genes, represented by 36 phyla.RESULTS: Truncation to 100 nucleotides (nt) downstream from the position corresponding to base 28 in the Escherichia coli 165 rRNA gene caused misclassification of 87 (39.7%) of the 219 sequences, compared with misclassification of only 29 (13.2%) sequences with truncation to 350 nt. Among 350-nt sequence reads within various regions of the 165 rRNA gene, the reverse read of an amplicon generated using the 343F/798R primers had the least (8.2%) effect on classification. In comparison, truncation to 900 nt mimicking single pass Sanger reads misclassified 5.0% of the 219 sequences. The 343F/798R amplicon accurately assigned 91.8% of the 219 sequences at the species level. Weighted by abundance of the species in the esophageal dataset, the 343F/798R amplicon yielded similar classification accuracy without a significant loss in species coverage (92%). Modification of the 343F/798R primers to 347F/803R increased their universality among foregut species. Assuming that a typical polymerase chain reaction can tolerate 2 mismatches between a primer and a template, the modified 347F and 803R primers should be able to anneal 98% and 99.6% of all 16S rRNA genes in the RDP database.CONCLUSION: 347F/803R is the most suitable pair of primers for classification of foregut 16S rRNA genes but also possess universality suitable for analyses of other complex microbionnes. (C) 2010 Baishideng. All rights reserved.